INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 1V2i?5JU ID3907 •S7 Dlebor, Samuel, 1926- 1952 effects of various ontimetaboliles on the growth and development of F.nna plpiens. 70p. illu s ., diegrs., tables. Thesic (:h ,1.; - N.Y.lr. , Graduate School, 19^2. Bibliography: p,/j3~-'r0« C98303 Shelf Ust Xerox University Microfilms, Ann Arbor, Michigan 48106 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. .MHfUXI % rrM'’^ v :n THE EFFECTS OF VARIOUS ANTIMETABOLITES ON THE GROWTH AND DEVELOPMENT OF RANA PIPIENS Samuel Bieber A pril 1958 A D issertation in: the Department of Biology Submitted in P a rtia l F ulfillm ent of the Requirements fo r the Degree of Doctor of Philosophy at New York U niversity TABLE OF CONTENTS Acknowledgments. Introduction Pages 1-5 M aterials and Methods Pages 6-10 Results: Introduction Pages 11-13 I.. Comparative A ctivity of the Analogues Pages 13-16 II* Growth Patterns as Re­ vealed by A ntim etabolites Pages 16-19 III* Reversal Experiments Pages 19-31 m . M orphological R esults A* Gross Pages 21-23 B. H istological Pages 23-27 D iscussion Pages 28-39 Summary Pages 40-42 Bibliography Pages 43-48 Appendix I Tables I-III Pages 49-51 Plates 1-IX Pages 52-60 (30 Figures) Appendix II Data used in constructing graphs (Figures 1-8) Pages 61-68 Appendix III Source® o f H feterials Pages 69-70 3.0 I ^ ACKNOWLEDGMENTS I would lik e to express my sincere appreciation to Dr. Ross. F. N ig relli, under whose sponsorship th is in v estig atio n was carried out. The major portion of th is investig atio n was carried out under a Burroughs-Wellcome Research Fellowship jo in tly sponsored by New York U niversity and the New York Zoolo­ g ical Society. I am g ratefu l to Dr. Harry A. Charipper fo r making the fa c ilitie s of the Biology Department of Washington Square College available to me and fo r acting as adm inistrator of the Research Fellow ship. I am indebted to Dr. George H. H itchings of the Wellcome Research L aboratories fo r h is many suggestions concerning the problem and the m anuscript and fo r sup­ plying generous q u an tities of the analogues and metabo­ lite s used in these stu d ies. The am inopterin, fo lic acid and leucovorin used in th is investigation were graciously supplied by Dr. Ruegsegger of the Lederle L aboratories a t P earl River, New York. I would also lik e to thank Dr. Seymour Hutner of the Haskins L aboratories and Dr* E li Goldsmith of the College of D entistry for th e ir encouragement and con­ stru ctiv e criticism . The photomicrographs were prepared by Mr.. A lbert Stenger of the Biology Department of Washington Square College. Last but not le a st I would lik e to thank my wife Rosalyn fo r her aid in the preparation of th is manu­ sc rip t. - I - INTRODUCTION E hrlich, in the Harben Lectures of 1907 (13), was the f ir s t to present ideas sim ilar to the present-day concept of antim etabolites. While E h rlich 's views were speculative, subsequent studies of enzyme actio n , bacter­ ia l and animal n u tritio n , and studies of the pharmaco­ lo g ical antagonisms of drugs have allowed the form ula­ tio n of a concept of antim etabolites in more precise term s. The a b ility of a substance, stru c tu ra lly resemb­ ling one of the components of a reactiv e system , to in ­ h ib it the reaction was recognized by enzym ologists in ■ the early 1930's. The lite ra tu re of the related field s of biochem istry and biology* in the period from 1920 to 1940, revealed several instances of chance observa­ tions th at stru ctu ral analogues of a m etabolite In te r­ fe re d w ith its biological a c tiv ity . Such findings, remarkable as they were, were frequently branded as erro rs. I t w asn't u n til the rep o rt of Woods in 1940 (53) th at the biochemical im plications were realized . He reported th at the b a cterio static actio n of sulfanilam ide and related sulfonamide drugs was com petitively reversed by p-aminabenzoic acid (PABA). PABA had not previously been recognized as a bio lo g ically im portant compound, but Woods' prediction was soon v e rified . The sulfonamide drugs were thus viewed as stru ctu ral analogues of th is, m etabolite and were said to owe th e ir b a cterio static * 2 • properties to th is fa c t. I t was in ferred th a t they acted by producing a deficiency, in the b acterial c e ll, of th is esse n tia l su b strate, w ithout which the organism was un­ able to m ultiply. The rep o rt of Woods prompted a widespread search fo r chem otherapeutic agents among compounds stru c tu ra lly related to m etabolites.. Since then the fie ld of in h ib i­ tio n analysis has received tremendous impetus from the synthesis of many such compounds. I t has also come to include those compounds which, though not stru ctu rally analogous to m etabolites, com petitively in h ib it th eir u tiliz a tio n by an organism* Almost a ll of the pioneer work in the fie ld has been carried .o u t on b acteria. Interference w ith the u tiliz a tio n of th e ir m etabolites was read ily reflected in an in h ib itio n of growth which could be measured. Such studies have shed lig h t on many of the in tric a c ie s of biochem ical mechanisms. Studies carried out on higher forms have been prim arily concerned w ith the e ffe cts on organ system s. Recently a few re­ ports appeared concerning investigations carried out on the developing chick (1, 8, 9, 31,; 48,, 52, 54). Aside from the gross e ffe cts on survival and attem pts a t meta­ bolic rev ersal of in h ib itio n , these in v estig ato rs did not attem pt to observe the more subtle effects on growth and on the d iffe re n tia tio n of the various organ system a, or possibly on the in itia tio n of the various physiological or biochem ical phenomena. Except fo r a b rie f attem pt by Brachet (6) and a more recent prelim inary report by Rosenbaum and Verlardo (40), amphibia have not been u ti­ lized fo r such stu d ies. The amphibian egg lends its e lf read ily to experim ental and biochem ical observations on early morphogenesis. The chick embryo is not easily ac­ cessible for chemical studies before the second or th ird day of incubation, by which time early m orphogenesis is ended. H itchings and his collaborators have made an in ten ­ sive study of the in h ib itio n of the growth of Lacto­ b acillu s easel and other systems by analogues of purine and pyrim idine bases (5, 7, 14-19, 25-28, 42, 48). Over a period of ten years several hundred such analogues were synthesized and tested . They were found to have a v ariety of biological effects which were attrib u ted to various types and degrees of interference w ith the biosynthesis of nucleic acids and related compounds. The key posi­ tio n of nucleic acids in the fundamental processes of growth and d iffe re n tia tio n has been w ell established. Thus the d e sira b ility of testin g such antim etabolites on a developing form is obvious. Such studies might co n tri­ bute to the comparative biochem istry of nucleic acid metabolism and to the knowledge of the mode of action of the antim etabolites. These compounds might also provide some knowledge as to the relatio n sh ip of sp ecific compounds - 4 - to morphologic development. I t was the consideration of the preceding facts th at led to undertaking the follow ing study of antim etabolites and amphibian development. The leopard frog, Rana p iu ien s. was chosen because its re la tiv e ly short period of develop-* ment affords the opportunity to study the aforem entioned phenomena. A prelim inary attem pt at such studies was undertaken by B ieber, N ig relli and H itchings (4 ). This study revealed th at despite the d iffic u ltie s inherent in immersion techniques careful experim ental m anipulation makes the approach fe a sib le . Along w ith the purine and pyrim idine analogues, am lnopterin (4-am inopteroylglutam ic acid) was used as a reference compound. The action of am inopterin in in te rfe rrin g w ith nucleic acid synthesis has been established in recent years (81, 45). This com­ pound had previously been tested on the developing chick (9, 31, 46, 58) and in a prelim inary fashion on the developing frog (40). As a re su lt of the prelim inary experim ent, the fo l­ lowing long range p ro ject fo r the study of antim etabolites on amphibian development was. se t up in conjunction w ith the Wellcome Research Foundation. In cases where success­ fu l In h ib itio n of development was obtained the follow ing experim ental procedures were to be applied. 1. In itia tio n of treatm ent a t various stages of development from fe rtiliz a tio n , through metamorphosis.